The simultaneous concentration and recovery of microbes in drinking water is a critical procedure for responding to potential water-related bioterrorism events, and also would be an important technique for cost-effective routine monitoring of drinking water quality. Simultaneous microbe recovery can be accomplished in large-volume (100+L) water samples using ultrafiltration (hereinafter “UF”), although little published research is available that indicates what process conditions are effective for yielding high recovery efficiencies for viruses, bacteria, and parasites in a single water sample.
Ultrafiltration is a technique that can be utilized for the simultaneous concentration of water-borne microbes but is more readily known by the public as a medical technique (hemodialysis) for people with kidney failure, where ultrafilter “dialyzers” are used to mimic the filtration activity of the kidneys by filtering blood to remove excess water, salts and waste products while retaining blood cells and proteins. Ultrafilters have pore sizes small enough to separate particles from water, as well as molecules that are larger than the Molecular Weight Cut-Off (hereinafter “MWCO”) of the ultrafilter. Ultrafilters typically have MWCOs in the 10,000-100,000 Dalton (“Da”) range (i.e. 10 kDa-100 kDa). Molecules smaller than the MWCO, therefore, such as water molecules, salts and small organic compounds, will simply pass through an ultrafilter as “permeate” and will not be co-concentrated with larger molecules and particles.
Ultrafiltration has been investigated since the 1970s as a technique for the concentration of microbes in drinking water (Belfort, G., Rotem, Y., Katzenelson, E., “Virus concentration using hollow fiber membranes-ii.” Water Research, 1976, v. 10(4): pp. 279-284). In the early 1980s, tangential-flow hollow fiber UF was investigated and found to be effective for recovering viruses in large-volume (up to 100-L) of tap water samples (Dziewulski, D. M., Belfort, G., “Virus concentration from water using high-rate tangential-flow hollow fiber ultrafiltration,” Water Science and Technology, 1983 v. 15:75-89). More recently, research reported greater than 50% recoveries for bacteriophages, E. coli and C. parvum oocysts seeded into 10-L surface water samples (Morales-Morales, H., Vidal, G., Olszewski, J., Rock, C., Dasgupta, D., Oshima, K., Smith, G., “Optimization of a reusable hollow-fiber ultrafilter for simultaneous concentration of enteric bacteria, protozoa, and viruses from water,” Applied Environmental Microbiology, 2003, v. 69(7): pp. 4098-4102). While the simultaneous UF recovery results of Morales-Morales et al. were good, their technique relied on the use of a calf serum protocol to pre-treat the ultrafilter membranes prior to filtration. For certain applications (e.g., rapid response, and field-based filtration), pre-treatment with calf serum may not be appropriate or practical due to the potential for contaminating microbial growth in filters pre-treated with calf serum.
Research conducted at the Centers for Disease Control and Prevention, National Center for Infectious Diseases (hereinafter “CDC”) has shown that UF can be an effective technique for simultaneously concentrating viruses, bacteria, and parasites in 100 L samples of drinking water (Hill, V. R., Polaczyk A. L., Hahn D., Jothikumar N., Cromeans T. L., Roberts J. M., Amburgey J. E. “Development of a rapid method for simultaneously recovering microbes in drinking water using ultrafiltration with sodium polyphosphate and surfactants.” Applied Environmental Microbiology, 2005, 71(11):6878-6884). Ultrafilters that can accommodate 100 L water samples at practical process times have holdup volumes that are at best 250 mL or more; these volumes are too large for sensitive molecular or immunological detection of pathogens. Therefore, although it is likely that a UF procedure can be effective for simultaneous microbe recovery, it is unlikely that UF techniques are capable of reducing sample volumes to levels (<10 mL) sufficient for detecting low concentrations of microbes in a water sample and/or screening of the separated microbes based on specific conductivity and size.
In contrast, insulator-based dielectrophoresis (hereinafter “iDEP”) systems are known to be capable of capturing, concentrating, and separating microbes in very small (<1 to 10 mL) water samples. Cummings and Singh have demonstrated iDEP separation and trapping with polystyrene particles using DC electric fields and a variety of arrays of insulating posts (Cummings, E., Singh, A., “Dielectrophoretic trapping without embedded electrodes,” SPIE: Conference on Microfluidic Devices and Systems III, 2000, Santa Clara, Calif., Proc. SPIE, 4177: pp. 164-173). Chou et al., demonstrated iDEP trapping of DNA molecules, E. coli cells and blood cells using insulating structures and AC electric fields (Chou, C., Tegenfeldt, J., Bakajin, O., Chan, S., Cox, E., Darnton, N., Duke, T., Austin, R., “Electrodeless dielectrophoresis of single- and double-stranded DNA,” Biophysical Journal, 2002, v. 83(4): pp. 2170-2179). Zhou et al., and Suehiro et al., used a channel filled with insulating glass beads and AC electric fields for separating and concentrating yeast cells in water (Zhou, G., Imamura, M., Suehiro, J., Hara, M., “A dielectrophoretic filter for separation and collection of fine particles suspended in liquid,” 37th Annual Meeting of the IEEE-Industry-Applications-Society, 2002, Pittsburgh, Pa., Proc. IEEE: pp. 1404-1411; and Suehiro, J., Zhou, G., Imamura, M., Hara, M., “Dielectrophoretic filter for separation and recovery of biological cells in water,” IEEE Annual Meeting of the Industry-Applications-Society, 2003, Pittsburgh, Pa., Proc. IEEE, v. 39: pp. 1514-1521). Finally, Lapizco-Encinas, et al., have demonstrated the selective dielectrophoretic trapping and concentration of live and dead E. coli cells, the separation of four different species of live bacterial cells, and the concentration of spores and viruses in both glass and plastic chips (Lapizco-Encinas, B. H., Simmons, B. A., Cummings, E. B., Fintschenko, Y., “Dielectrophoretic concentration and separation of live and dead bacteria in an array of insulators,” Analytical Chemistry, 2004, v. 76(6): pp. 1571-1579; Lapizco-Encinas, B. H., Simmons, B. A., Cummings, E. B., Fintschenko, Y., “Insulator-based dielectrophoresis for the selective concentration and separation of live bacteria in water,” Electrophoresis, 2004, v. 25(10-11): pp. 1695-704; Lapizco-Encinas, B. H., Davalos, R., Simmons, B. A., Cummings, E. B., Fintschenko, Y., “An insulator-based (electrodeless) dielectrophoretic concentrator for microbes in water,” Journal of Microbiological Methods, 2005, v. 62(3), pp. 317-326; and Simmons, B. A., Lapizco-Encinas, B. H., Shediac, R., Hachman, J., Chames, J., Fiechtner, G., Cummings, E., Fintschenko, Y., “Polymeric insulating post electrodeless dielectrophoresis (iDEP) for the monitoring of water-borne pathogens,” The 8th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 2004, Malmo, Sweden, Royal Society of Chemistry Special Publication, 2005, v. 297: pp. 171-173).
The combination of UF with iDEP, therefore, holds potential promise for allowing water utilities and associated industries to accurately assess low levels of pathogens in finished drinking water samples, whether due to natural or intentional contamination. This approach also could be applied to monitoring source water, industrial effluent, hospital discharge, and military water infrastructures for pathogens. Moreover, iDEP technology can separate live from dead/damaged microbes, thereby decreasing the chances of generating false-positive PCR results due to the presence of naked nucleic acid or non-viable microbes. In addition, the iDEP technique has the potential for sorting microbes according to type (e.g., viruses, bacteria, and parasites).